We reported previously that insect acetylcholinesterases (AChEs) could be selectively and irreversibly inhibited by methanethiosulfonates presumably through conjugation to an insect-specific cysteine in these enzymes. However, no direct proof for the conjugation has been published to date, and doubts remain about whether such cysteine-targeting inhibitors have desirable kinetic properties for insecticide use. Here we report mass spectrometric proof of the conjugation and new chemicals that irreversibly inhibited African malaria mosquito AChE with bimolecular inhibition rate constants (kinact/KI) of 3,604–458,597 M−1sec−1 but spared human AChE. In comparison, the insecticide paraoxon irreversibly inhibited mosquito and human AChEs with kinact/KI values of 1,915 and 1,507 M−1sec−1, respectively, under the same assay conditions. These results further support our hypothesis that the insect-specific AChE cysteine is a unique and unexplored target to develop new insecticides with reduced insecticide resistance and low toxicity to mammals, fish, and birds for the control of mosquito-borne diseases.